RESUMEN
Gut microbiome targeting has emerged as a new generation of personalized medicine and a potential wellness and disease driver. Specifically, the gut redox balance plays a key role in shaping the gut microbiota and its link with the host, immune system, and disease evolution. In this sense, precise and personalized nutrition has proven synergy and capability to modulate the gut microbiome environment through the formulation of dietary interventions, such as vitamin support. Accordingly, there are urgent demands for simple and effective analytical platforms for understanding the relationship between the tailored vitamin administration and the gut microbiota balance by rapid noninvasive on-the-spot oxidation/reduction potential monitoring for frequent and close surveillance of the gut redox status and targeting by personalized nutrition interventions. Herein, we present a disposable potentiometric sensor chip and a homemade multiwell potentiometric array to address the interplay of vitamin levels with the oxidation/reduction potential in human feces and saliva. The potentiometric ORP sensing platforms have been successfully validated and scaled up for the setup of a multiapplication prototype for cross-talk-free simple screening of many specimens. The interpersonal variability of the gut microbiota environment illustrates the potential of feces and saliva samples for noninvasive, frequent, and decentralized monitoring of the gut redox status to support timely human microbiota surveillance and guide precise dietary intervention toward restoring and promoting personalized gut redox balance.
Asunto(s)
Microbioma Gastrointestinal , Microbiota , Humanos , Heces , Vitaminas , Oxidación-ReducciónRESUMEN
BACKGROUND & AIMS: We analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis. METHODS: We obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD (43 with Crohn's disease [CD], 23 with ulcerative colitis [UC]), and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed. RESULTS: We found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents. CONCLUSIONS: We identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes.
Asunto(s)
Colitis Ulcerosa/genética , Colon Sigmoide/metabolismo , Enfermedad de Crohn/genética , Metilación de ADN , Epigénesis Genética , Células Epiteliales/metabolismo , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Transcripción Genética , Transcriptoma , Adolescente , Factores de Edad , Biopsia , Estudios de Casos y Controles , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/microbiología , Colon Sigmoide/microbiología , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/microbiología , Diagnóstico Diferencial , Inglaterra , Células Epiteliales/microbiología , Femenino , Microbioma Gastrointestinal , Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo , Humanos , Íleon/microbiología , Mucosa Intestinal/microbiología , Masculino , Organoides , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Ribotipificación , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Sarcoidosis is characterized by noncaseating granulomas with an unknown cause that present primarily in the lung. Propionibacterium acnes, an immunogenic commensal skin bacterium involved in acne vulgaris, has been implicated as a possible causative agent of sarcoidosis. Here, we demonstrate that a viable strain of P. acnes isolated from a patient with sarcoidosis and instilled intratracheally into wild-type mice can generate pulmonary granulomas similar to those observed in patients with sarcoidosis. The formation of these granulomas is dependent on the administration of viable P. acnes. We also found that mice deficient in the innate immunity adapter protein MyD88 had a greater number and a larger area of granuloma lesions compared with wild-type mice administered P. acnes. Early after P. acnes administration, wild-type mice produced proinflammatory mediators and recruited neutrophils into the lung, a response that is dependent on MyD88. In addition, there was an increase in granuloma number and size after instillation with P. acnes in mice deficient in CybB, a critical component of nicotinamide adenine dinucleotide phosphate oxidase required for the production of reactive oxygen species in the phagosome. Myd88-/- or Cybb-/- mice both had increased persistence of P. acnes in the lung, together with enhanced granuloma formation. In conclusion, we have generated a mouse model of early granuloma formation induced by a clinically relevant strain of P. acnes isolated from a patient with sarcoidosis, and, using this model, we have shown that a deficiency in MyD88 or CybB is associated with impaired bacterial clearance and increased granuloma formation in the lung.
Asunto(s)
Granuloma/metabolismo , Granuloma/microbiología , Pulmón/microbiología , Pulmón/patología , Glicoproteínas de Membrana/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , NADPH Oxidasas/metabolismo , Propionibacterium acnes/fisiología , Animales , Modelos Animales de Enfermedad , Granuloma/patología , Mediadores de Inflamación/metabolismo , Glicoproteínas de Membrana/deficiencia , Ratones Endogámicos C57BL , Viabilidad Microbiana , Factor 88 de Diferenciación Mieloide/deficiencia , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sarcoidosis Pulmonar/microbiología , Sarcoidosis Pulmonar/patología , Tráquea/microbiologíaRESUMEN
BACKGROUND: Propionibacteria are part of the human microbiota. Many studies have addressed the predominant colonizer of sebaceous follicles of the skin, Propionibacterium acnes, and investigated its association with the skin disorder acne vulgaris, and lately with prostate cancer. Much less is known about two other propionibacterial species frequently found on human tissue sites, Propionibacterium granulosum and Propionibacterium avidum. Here we analyzed two and three genomes of P. granulosum and P. avidum, respectively, and compared them to two genomes of P. acnes; we further highlight differences among the three cutaneous species with proteomic and microscopy approaches. RESULTS: Electron and atomic force microscopy revealed an exopolysaccharide (EPS)-like structure surrounding P. avidum cells, that is absent in P. acnes and P. granulosum. In contrast, P. granulosum possesses pili-like appendices, which was confirmed by surface proteome analysis. The corresponding genes were identified; they are clustered with genes encoding sortases. Both, P. granulosum and P. avidum lack surface or secreted proteins for predicted host-interacting factors of P. acnes, including several CAMP factors, sialidases, dermatan-sulphate adhesins, hyaluronidase and a SH3 domain-containing lipoprotein; accordingly, only P. acnes exhibits neuraminidase and hyaluronidase activities. These functions are encoded on previously unrecognized island-like regions in the genome of P. acnes. CONCLUSIONS: Despite their omnipresence on human skin little is known about the role of cutaneous propionibacteria. All three species are associated with a variety of diseases, including postoperative and device-related abscesses and infections. We showed that the three organisms have evolved distinct features to interact with their human host. Whereas P. avidum and P. granulosum produce an EPS-like surface structure and pili-like appendices, respectively, P. acnes possesses a number of unique surface-exposed proteins with host-interacting properties. The different surface properties of the three cutaneous propionibacteria are likely to determine their colonizing ability and pathogenic potential on the skin and at non-skin sites.
Asunto(s)
Hibridación Genómica Comparativa , Genoma Bacteriano , Interacciones Huésped-Patógeno/genética , Propionibacterium/genética , ADN Bacteriano/genética , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Propionibacterium/citología , Propionibacterium/ultraestructura , Análisis de Secuencia de ADN , Piel/microbiologíaRESUMEN
BACKGROUND: Inflammation is commonly observed in radical prostatectomy specimens, and evidence suggests that inflammation may contribute to prostate carcinogenesis. Multiple microorganisms have been implicated in serving as a stimulus for prostatic inflammation. The pro-inflammatory anaerobe, Propionibacterium acnes, is ubiquitously found on human skin and is associated with the skin disease acne vulgaris. Recent studies have shown that P. acnes can be detected in prostatectomy specimens by bacterial culture or by culture-independent molecular techniques. METHODS: Radical prostatectomy tissue samples were obtained from 30 prostate cancer patients and subject to both aerobic and anaerobic culture. Cultured species were identified by 16S rDNA gene sequencing. Propionibacterium acnes isolates were typed using multilocus sequence typing (MLST). RESULTS: Our study confirmed that P. acnes can be readily cultured from prostatectomy tissues (7 of 30 cases, 23%). In some cases, multiple isolates of P. acnes were cultured as well as other Propionibacterium species, such as P. granulosum and P. avidum. Overall, 9 of 30 cases (30%) were positive for Propionibacterium spp. MLST analyses identified eight different sequence types (STs) among prostate-derived P. acnes isolates. These STs belong to two clonal complexes, namely CC36 (type I-2) and CC53/60 (type II), or are CC53/60-related singletons. CONCLUSIONS: MLST typing results indicated that prostate-derived P. acnes isolates do not fall within the typical skin/acne STs, but rather are characteristic of STs associated with opportunistic infections and/or urethral flora. The MLST typing results argue against the likelihood that prostatectomy-derived P. acnes isolates represent contamination from skin flora.
Asunto(s)
Infecciones por Bacterias Grampositivas/microbiología , Tipificación de Secuencias Multilocus/métodos , Propionibacterium acnes/aislamiento & purificación , Propionibacterium/aislamiento & purificación , Próstata/microbiología , Neoplasias de la Próstata/microbiología , Adulto , Anciano , Humanos , Masculino , Persona de Mediana Edad , Propionibacterium/genética , Propionibacterium acnes/genética , Prostatectomía , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Prostatic inflammation has been linked to a number of prostatic diseases such as benign prostatic hyperplasia (BPH), prostatitis syndromes, and prostate cancer. Major unanswered questions include what pathogenic mechanisms, such as bacterial infections, may drive the accumulation of inflammatory infiltrates in the human prostate, and how inflammation might contribute to disease. To study this potential link in an in vivo system, we developed a mouse model of long-term bacteria-induced chronic inflammation of the prostate using a human prostatectomy-derived strain of Propionibacterium acnes. METHODS: C57BL/6J mice were inoculated, via urethral catheterization, with vehicle control or a prostatectomy-derived strain of P. acnes (PA2). Animals were assessed at 2 days, 1, 2, or 8 weeks post-inoculation via histology and immunohistochemistry (IHC). RESULTS: PA2 inoculation resulted in severe acute and chronic inflammation confined to the dorsal lobe of the prostate. Chronic inflammation persisted for at least 8 weeks post-inoculation. Inflammatory lesions were associated with an increase in the Ki-67 proliferative index, and diminished Nkx3.1 and androgen receptor (AR) production. Interestingly, the observed response required live bacteria and both IHC and in situ hybridization assays for P. acnes indicated a potential intracellular presence of P. acnes in prostate epithelial cells. CONCLUSIONS: To our knowledge, this is the first mouse model of long-term prostatic inflammation induced by P. acnes, and more generally, any prostatectomy-derived bacterial isolate. This model may serve as a valuable preclinical model of chronic prostatic inflammation that can be used to mechanistically study the link between inflammation and prostatic disease.
Asunto(s)
Modelos Animales de Enfermedad , Propionibacterium acnes/crecimiento & desarrollo , Neoplasias de la Próstata/microbiología , Prostatitis/microbiología , Animales , Enfermedad Crónica , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Propionibacterium acnes/aislamiento & purificación , Prostatectomía , Neoplasias de la Próstata/cirugíaRESUMEN
The contribution of the human microbiota to health and disease is poorly understood. Propionibacterium acnes is a prominent member of the skin microbiota, but is also associated with acne vulgaris. This bacterium has gained recent attention as a potential opportunistic pathogen at non-skin infection sites due to its association with chronic pathologies and its isolation from diseased prostates. We performed comparative global-transcriptional analyses for P. acnes infection of keratinocytes and prostate cells. P. acnes induced an acute, transient transcriptional inflammatory response in keratinocytes, whereas this response was delayed and sustained in prostate cells. We found that P. acnes invaded prostate epithelial cells, but not keratinocytes, and was detectable intracellularly 7 days post infection. Further characterization of the host cell response to infection revealed that vimentin was a key determinant for P. acnes invasion in prostate cells. siRNA-mediated knock-down of vimentin in prostate cellsattenuated bacterial invasion and the inflammatory response to infection. We conclude that host cell tropism, which may depend on the host protein vimentin, is relevant for P. acnes invasion and in part determines its sustained inflammatory capacity and persistence of infection.
Asunto(s)
Endocitosis , Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Inflamación , Propionibacterium acnes/patogenicidad , Vimentina/metabolismo , Acné Vulgar/microbiología , Acné Vulgar/patología , Línea Celular , Células Epiteliales/inmunología , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Prostatitis/microbiología , Prostatitis/patología , Vimentina/genéticaRESUMEN
Lactic acid bacteria produce γ-aminobutyric acid (GABA) as an acid stress response. GABA is a neurotransmitter that may improve sleep and resilience to mental stress. This study focused on the selection, identification and optimization of a bacterial strain with high GABA production, for development as a probiotic supplement. The scientific literature and an industry database were searched for probiotics and potential GABA producers. In silico screening was conducted to identify genes involved in GABA production. Subsequently, 17 candidates were screened for in vitro GABA production using thin layer chromatography, which identified three candidate probiotic strains Levilactobacillus brevis DSM 20054, Lactococcus lactis DS75843and Bifidobacterium adolescentis DSM 24849 as producing GABA. Two biosensors capable of detecting GABA were developed: 1. a transcription factor-based biosensor characterized by the interaction with the transcriptional regulator GabR was developed in Corynebacterium glutamicum; and 2. a growth factor-based biosensor was built in Escherichia coli, which used auxotrophic complementation by expressing 4-aminobutyrate transaminase (GABA-T) that transfers the GABA amino group to pyruvate, hereby forming alanine. Consequently, the feasibility of developing a workflow based on co-culture with producer strains and a biosensor was tested. The three GABA producers were identified and the biosensors were encapsulated in nanoliter reactors (NLRs) as alginate beads in defined gut-like conditions. The E. coli growth factor-based biosensor was able to detect changes in GABA concentrations in liquid culture and under gut-like conditions. L. brevis and L. lactis were successfully encapsulated in the NLRs and showed growth under miniaturized intestinal conditions.
Asunto(s)
Lactobacillales , Lactobacillales/genética , Flujo de Trabajo , Escherichia coli/genética , 4-Aminobutirato Transaminasa , AlaninaRESUMEN
Prostate cancer (PCa) is the second leading cause of male cancer deaths in the Western world. Mounting evidence has revealed that chronic inflammation can be an important initiating factor of PCa. Recent work has detected the anaerobic Gram-positive bacterium Propionibacterium acnes in cancerous prostates, but with wide-ranging detection rates. Here, using in situ immunofluorescence (ISIF), P. acnes was found in 58 out of 71 (81.7%) tested cancerous prostate tissue samples, but was absent from healthy prostate tissues (20 samples) and other cancerous tissue biopsies (59 mamma carcinoma samples). Live P. acnes bacteria were isolated from cancerous prostates and cocultured with the prostate epithelial cell line RWPE1. Microarray analysis showed that the host cell responded to P. acnes with a strong multifaceted inflammatory response. Active secretion of cytokines and chemokines, such as IL-6 and IL-8, from infected cells was confirmed. The host cell response was likely mediated by the transcriptional factors NF-κB and STAT3, which were both activated upon P. acnes infection. The P. acnes-induced host cell response also included the activation of the COX2-prostaglandin, and the plasminogen-matrix metalloproteinase pathways. Long-term exposure to P. acnes altered cell proliferation, and enabled anchorage-independent growth of infected epithelial cells, thus initiating cellular transformation. Our results suggest that P. acnes infection could be a contributing factor to the initiation or progression of PCa.
Asunto(s)
Células Epiteliales/microbiología , Infecciones por Bacterias Grampositivas/complicaciones , Infecciones por Bacterias Grampositivas/microbiología , Propionibacterium acnes/patogenicidad , Neoplasias de la Próstata/microbiología , Prostatitis/complicaciones , Prostatitis/microbiología , Anciano , Quimiocinas/biosíntesis , Células Epiteliales/inmunología , Perfilación de la Expresión Génica , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/patología , Humanos , Hibridación Fluorescente in Situ/métodos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Prevalencia , Propionibacterium acnes/aislamiento & purificación , Prostatitis/epidemiología , Prostatitis/patologíaRESUMEN
BACKGROUND: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. RESULTS: Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, ß-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. CONCLUSIONS: Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates. Thus, our data presents a rich resource for guiding much-needed investigations on P. acnes virulence factors and host interacting properties.
Asunto(s)
Acné Vulgar/microbiología , Proteínas Bacterianas/metabolismo , Propionibacterium acnes/metabolismo , Proteómica , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Electroforesis en Gel Bidimensional , Humanos , Datos de Secuencia Molecular , Propionibacterium acnes/química , Propionibacterium acnes/genética , Propionibacterium acnes/aislamiento & purificación , Transporte de Proteínas , Alineación de SecuenciaRESUMEN
OBJECTIVES: The present study aimed to identify proximal and distal factors associated with real life community functioning among men with schizophrenia. Real life community functioning was defined to include: independent living, occupational functioning, social and leisure activities, and substance misuse, self-harm, and aggressive behaviour. METHOD: 225 men with schizophrenia or schizo-affective disorder were assessed at discharge from hospital and at six-monthly intervals during a two-year period. Information was available from structured interviews with the participants, family members and treatment staff, records of psychiatric treatment, social service files, and official criminal records. Symptoms were assessed using the Positive and Negative Symptom Scale and the Hamilton Rating Scale for Depression. Substance misuse was measured by self-report and hair and urine analyses. Predictors of outcome were divided into four categories: childhood, adulthood, year prior to functional outcome assessment, and con-current. RESULTS: In a final regression model, five predictors were significantly associated with patients' real life functioning: two adult life-time variables-a diagnosis of drug abuse/dependence and level of education and three current variables-victimisation, depression, and medication non-compliance. Most of the variance in the final outcome scores was explained by current predictors. DISCUSSION: In order to elevate levels of psychosocial functioning among men with schizophrenia, clinicians need to assess victimisation experiences, and, when present, design and implement interventions to help patients protect themselves. In addition, depression needs to be identified and treated, and compliance with antipsychotic medication assured.
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Víctimas de Crimen/psicología , Trastorno Depresivo/diagnóstico , Trastorno Depresivo/psicología , Esquizofrenia/diagnóstico , Psicología del Esquizofrénico , Ajuste Social , Actividades Cotidianas/psicología , Adulto , Agresión/psicología , Antipsicóticos/uso terapéutico , Víctimas de Crimen/estadística & datos numéricos , Comparación Transcultural , Trastorno Depresivo/epidemiología , Humanos , Masculino , Cumplimiento de la Medicación , Probabilidad , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Calidad de Vida , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/epidemiología , Conducta Autodestructiva/diagnóstico , Conducta Autodestructiva/epidemiología , Índice de Severidad de la Enfermedad , Trastornos Relacionados con Sustancias/diagnóstico , Trastornos Relacionados con Sustancias/epidemiologíaRESUMEN
Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection.
RESUMEN
Propionibacterium acnes is a Gram-positive bacterium that is closely associated with various parts of the human body, in particular with sebaceous follicles of the skin. It has also been frequently isolated from diseased human prostates. Here, we report draft genome sequences of two P. acnes strains, P6 and PA2, isolated from radical prostatectomy specimens.
RESUMEN
Propionibacterium acnes is a Gram-positive bacterium that colonizes various niches of the human body, particularly the sebaceous follicles of the skin. Over the last years a role of this common skin bacterium as an opportunistic pathogen has been explored. Persistence of P. acnes in host tissue has been associated with chronic inflammation and disease development, for example, in prostate pathologies. This study investigated the intracellular fate of P. acnes in macrophages after phagocytosis. In a mouse model of P. acnes-induced chronic prostatic inflammation, the bacterium could be detected in prostate-infiltrating macrophages at 2 weeks postinfection. Further studies performed in the human macrophage cell line THP-1 revealed intracellular survival and persistence of P. acnes but no intracellular replication or escape from the host cell. Confocal analyses of phagosome acidification and maturation were performed. Acidification of P. acnes-containing phagosomes was observed at 6 h postinfection but then lost again, indicative of cytosolic escape of P. acnes or intraphagosomal pH neutralization. No colocalization with the lysosomal markers LAMP1 and cathepsin D was observed, implying that the P. acnes-containing phagosome does not fuse with lysosomes. Our findings give first insights into the intracellular fate of P. acnes; its persistency is likely to be important for the development of P. acnes-associated inflammatory diseases.
Asunto(s)
Espacio Intracelular/microbiología , Macrófagos/microbiología , Propionibacterium acnes/fisiología , Ácidos/metabolismo , Animales , Biomarcadores/metabolismo , Catepsina D/metabolismo , Muerte Celular , Línea Celular , Endosomas/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/patología , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana , Fagosomas/metabolismo , Propionibacterium acnes/crecimiento & desarrollo , Propionibacterium acnes/aislamiento & purificación , Próstata/microbiología , Próstata/patología , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
P. acnes is a skin commensal that is frequently associated with inflammatory diseases such as acne vulgaris. Despite the availability of the genome sequence functional studies on P. acnes are scarce due to a lack of methods for genetic manipulation of this bacterium. Here we present an insertional mutagenesis approach for the inactivation of specific P. acnes genes. The gene of interest can be disrupted and replaced with an erythromycin-resistance cassette by employing homologous recombination. We used this method to generate knock-out mutants of camp2 (PPA0687) and camp4 (PPA1231), encoding CAMP factor homologs with predicted co-hemolytic activities. The successful inactivation of the two genes was confirmed by PCR and Western blotting experiments using specific anti-CAMP2/CAMP4 sera. The Δcamp2 but not the Δcamp4 mutant exhibited reduced hemolytic activity in the CAMP reaction with sheep erythrocytes, indicating that CAMP2 is the major active co-hemolytic factor of P. acnes. The biological relevance of the CAMP factors remains unclear as disruption of camp2 or camp4 did not significantly alter the transcriptome response of HaCaT cells to P. acnes. The here presented insertional mutagenesis approach will facilitate future studies on P. acnes.
Asunto(s)
Proteínas Bacterianas/genética , Técnicas de Inactivación de Genes/métodos , Proteínas Hemolisinas/genética , Mutagénesis Insercional/métodos , Propionibacterium acnes/genética , Animales , Antibacterianos/farmacología , Western Blotting , Eritrocitos/efectos de los fármacos , Eritromicina/farmacología , Hemólisis , Ratones , Reacción en Cadena de la Polimerasa , Recombinación Genética , Selección Genética , OvinosRESUMEN
Post-translational modifications of core histones are important components of the epigenetic landscape. Recent investigations of bacterial or toxin-induced effects on histone phosphorylation and acetylation in host cells have linked the changes to transcriptional alterations of key cellular response pathways. However, these changes may have other reasons and functional consequences. Here, we show that infection of gastric epithelial cell lines with the carcinogenic bacterium Helicobacter pylori leads to changes in histone H3 phosphorylation: type IV secretion system (T4SS)-dependent decreases of H3 phosphorylation levels at serine 10 (pH3Ser10) and threonine 3 (pH3Thr3) were observed. Immunofluorescence experiments with pH3Ser10 and cyclin B1 revealed that a H. pylori-induced transient pre-mitotic arrest was responsible for the observed reduction. This causal link was substantiated further by showing that H. pylori causes a strong decrease of the cell division cycle 25 (CDC25C) phosphatase. As a consequence, mitotic histone H3 kinases such as vaccinia-related kinase 1 (VRK1) and Aurora B were not fully activated in infected cells. We show that VRK1 activity, measured using a kinase activity assay, was reduced after H. pylori infection by approximately 40%. Moreover, overexpression of VRK1, but not Aurora B, compensated for the H. pylori-induced decrease of pH3Ser10. Rephosphorylation of H3Ser10 was IkappaB kinase alpha (IKKalpha)-dependent and occurred at later time points of infection. Taken together, our work highlights the impact of bacterial pathogens on host cell chromatin; this modulation reflects the subversion of key cellular processes such as cell cycle progression.